Granulocyte-macrophage Colony-stimulating Factor Expression Research Articles

Paracrine Activation of STAT3 Drives GM-CSF Expression in Breast Carcinoma Cells, Generating a Symbiotic Signaling Network with Breast Carcinoma-Associated Fibroblasts.

This study evaluated the paracrine signaling between breast carcinoma-associated fibroblasts (CAFs) and breast cancer (BCa) cells. Resolving cell-cell communication in the BCa tumor microenvironment (TME) will aid the development of new therapeutics. Here, we utilized our patented TAME (tissue architecture and microenvironment engineering) 3D culture microphysiological system, which is a suitable pathomimetic avatar for the study of the BCa TME. We cultured in 3D BCa cells and CAFs either alone or together in cocultures and found that when cocultured, CAFs enhanced the invasive characteristics of tumor cells, as shown by increased proliferation and spread of tumor cells into the surrounding matrix. Secretome analysis from 3D cultures revealed a relatively high secretion of IL-6 by CAFs. A marked increase in the secretion of granulocyte macrophage-colony stimulating factor (GM-CSF) when carcinoma cells and CAFs were in coculture was also observed. We theorized that the CAF-secreted IL-6 functions in a paracrine manner to induce GM-CSF expression and secretion from carcinoma cells. This was confirmed by evaluating the activation of STAT3 and gene expression of GM-CSF in carcinoma cells exposed to CAF-conditioned media (CAF-CM). In addition, the treatment of CAFs with BCa cell-CM yielded a brief upregulation of GM-CSF followed by a marked decrease, indicating a tightly regulated control of GM-CSF in CAFs. Secretion of IL-6 from CAFs drives the activation of STAT3 in BCa cells, which in turn drives the expression and secretion of GM-CSF. As a result, CAFs exposed to BCa cell-secreted GM-CSF upregulate inflammation-associated genes such as IL-6, IL-6R and IL-8, thereby forming a positive feedback loop. We propose that the tight regulation of GM-CSF in CAFs may be a novel regulatory pathway to target for disrupting the CAF:BCa cell symbiotic relationship. These data provide yet another piece of the cell-cell communication network governing the BCa TME.

Inhibition of non-small cell lung cancer metastasis by knocking down APE1 through regulating myeloid-derived suppressor cells-induced immune disorders.

Non-small cell lung cancer (NSCLC) represents a highly immunogenic malignancy. Immunologic tolerance facilitated by myeloid-derived suppressor cells (MDSCs) is implicated in primary or secondary resistance mechanisms in NSCLC. The potential role of APE1 in regulating NSCLC metastasis by targeting MDSCs remains uncertain. This study utilized a plasmid, Plxpsp-mGM-CSF, to induce elevated granulocyte-macrophage colony-stimulating factor (GM-CSF) expression in A549 cells. Tumor transplantation experiments involved A549, A549+GM-CSF, and A549+GM-CSF-siAPE1 cell lines. Evaluation encompassed MDSCs, Treg cells, IgG, CD3, and CD8 levels. Notably, lung cancer tissues and cells displayed markedly reduced APE1 expression. siAPE1 transfection significantly curtailed tumor growth compared to the A549+GM-CSF group. APE1 knockdown orchestrated immune system modulation in lung tumor mice, characterized by diminished MDSCs but augmented Treg cells, IgG, CD3, and CD8. Additionally, APE1 knockdown led to reduced levels of pro-MDSC cytokines (HGF, CCL5, IL-6, CCL12) and a concurrent upregulation of the anti-MDSC cytokine IL-1ra. Furthermore, APE1 knockdown impeded cell viability in both A549 and H1650 cells. Transplantation of A549-GM-CSF amplified MDSC levels, fostering accelerated tumor growth, while mitigating MDSC levels through APE1 knockdown hindered tumor progression and alleviated inflammatory infiltration in lung cancer tissues. Strategies targeting the APE1/MDSC axis offer a promising approach for lung cancer prevention and treatment, presenting novel insights for NSCLC management.

Scutellaria baicalensis water decoction ameliorates lower respiratory tract infection by modulating respiratory microbiota

BackgroundThe pathogenesis of lower respiratory tract infections (LRTIs) has been demonstrated to be strongly associated with dysbiosis of respiratory microbiota. Scutellaria baicalensis, a traditional Chinese medicine, is widely used to treat respiratory infections. However, whether the therapeutic effect of S. baicalensis on LRTIs depends upon respiratory microbiota regulation is largely unclear. PurposeTo investigate the potential effect and mechanism of S. baicalensis on the respiratory microbiota of LRTI mice. MethodsA mouse model of LRTI was established using Klebsiella pneumoniae or Streptococcus pneumoniae. Antibiotic treatment was administered, and transplantation of respiratory microbiota was performed to deplete the respiratory microbiota of mice and recover the destroyed microbial community, respectively. High-performance liquid chromatography (HPLC) was used to determine and quantify the chemical components of S. baicalensis water decoction (SBWD). Pathological changes in lung tissues and the expressions of serum inflammatory cytokines, including interleukin-17A (IL-17A), granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α), were determined by hematoxylin and eosin (H&E) staining and enzyme-linked immunosorbent assay (ELISA), respectively. Quantitative real-time PCR (qRT-PCR) analysis was performed to detect the mRNA expression of GM-CSF. Metagenomic sequencing was performed to evaluate the effect of SBWD on the composition and function of the respiratory microbiota in LRTI mice. ResultsSeven main components, including scutellarin, baicalin, oroxylin A-7-O-β-d-glucuronide, wogonoside, baicalein, wogonin, and oroxylin A, were identified and their levels in SBWD were quantified. SBWD ameliorated pulmonary pathological injury and inflammatory responses in K. pneumoniae and S. pneumoniae-induced LRTI mice, as evidenced by the dose-dependent reductions in the levels of serum inflammatory cytokines, IL-6 and TNF-α. SBWD may exert a bidirectional regulatory effect on the host innate immune responses in LRTI mice and regulate the expressions of IL-17A and GM-CSF in a microbiota-dependent manner. K. pneumoniae infection but not S. pneumoniae infection led to dysbiosis in the respiratory microbiota, evident through disturbances in the taxonomic composition characterized by bacterial enrichment, including Proteobacteria, Enterobacteriaceae, and Klebsiella. K. pneumoniae and S. pneumoniae infection altered the bacterial functional profile of the respiratory microbiota, as indicated by increases in lipopolysaccharide biosynthesis, metabolic pathways, and carbohydrate metabolism. SBWD had a certain trend on the regulation of compositional disorders in the respiratory flora and modulated partial microbial functions embracing carbohydrate metabolism in K. pneumoniae-induced LRTI mice. ConclusionSBWD may exert an anti-infection effect on LRTI by targeting IL-17A and GM-CSF through respiratory microbiota regulation. The mechanism of S. baicalensis action on respiratory microbiota in LRTI treatment merits further investigation.

Abstract 5368: Targeting tumor-intrinsic S100A1 augments antitumour immunity and potentiates anti-PD-1 efficacy

Abstract Immune-checkpoint blockade (ICB) has revolutionized cancer treatment. However, only a minority of patients achieve the long-term, durable response to ICB, and the response rates in most advanced tumors is limited to 10%-25%. The unsatisfactory overall response to ICB and the great variation in response among individuals highlight the necessity for developing predictive biomarkers and overcoming ICB resistance. A potential mode of resistance is tumor-mediated immunosuppression leading to inhibition of T cell-mediated infiltration and immunity. Here, combining single-cell and bulk transcriptomic analysis with liquid biopsy of tumor patients, we showed that S100 calcium binding protein A1 (S100A1) was closely related to ICB response, and that detecting plasma S100A1 level could help predicting the response to ICB, implying that tumor-intrinsic S100A1 may contribute to immunosuppressive tumor microenvironment (TME) and resistance to ICB. Importantly, endogenous S100a1 knockdown (S100a1KD) promoted anti-tumor immunity, conferred tumor regression, and sensitized tumors to anti-PD-1 immunotherapy in multiple syngeneic murine models. By characterizing the immune landscape of S100a1KD syngeneic murine tumor models and non-small cell lung cancer (NSCLC) clinical samples, we demonstrated a negative relationship between tumor-intrinsic S100A1 expression and infiltration of M1-like macrophage and CD8+ T cells. Single cell and spatial transcriptomic analyses, together with functional studies, have revealed that ablation of tumor-intrinsic S100A1 exerts profound impacts on the M1-like polarization of macrophages and cytotoxicity of CD8+ T cells. Moreover, the conditioned medium from S100a1KD tumor cells promoted M1 polarization of both bone marrow-derived macrophages (BMDMs) and Raw264.7 cells, which in turn reinvigorated tumor-specific T cell immunity both in vitro and in vivo. Of note, as a secreted protein, introducing recombinant S100A1 into the medium from S100a1KD tumor cells could not reverse the effects on T cells and macrophages, suggesting that these effects were independent of extracelluar S100A1 secretion and that S100A1 may act as an intracellular signaling molecule. Mechanistically, S100A1 affects deubiquitination of NF-κB through interacting with the ubiquitin-like (Ubl) domains of ubiquitin specific protease 7 (USP7), leading to upregulated granulocyte-macrophage colony-stimulating factor (GM-CSF) expression. Collectively, these data identified the S100A1/USP7/p65/GM-CSF inhibitory axis as a critical immune suppressor on M1-like macrophage polarization and T cell immunity for the promotion of tumor progression and resistance to ICB. Our findings demonstrated the potential of plasma S100A1 expression in predicting the response to ICB, and provide a rationale for combining GM-CSF and anti-PD-1 immunotherapy in tumors with high S100A1 expression. Citation Format: Yufeng Guo, Hua Bai, Jie Wang. Targeting tumor-intrinsic S100A1 augments antitumour immunity and potentiates anti-PD-1 efficacy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5368.

Saharan dust induces the lung disease-related cytokines granulocyte–macrophage colony-stimulating factor and granulocyte colony-stimulating factor

Desert dust exposure is associated with adverse respiratory health effects. Desert dust is a complex pollutant mixtures that includes respirable crystalline and amorphous particles, metals, and microbial constituents. Given the health effects of desert dust and its heterogeneity, as yet unidentified harmful biological pathways may be triggered. Therefore, we exposed human in vitro air–liquid interface co-cultures of alveolar epithelial A549 cells and THP-1 macrophages to Saharan dust (SD). For comparison, we used the known pulmonary toxicant DQ12 quartz dust. Via RNA sequencing, we identified that SD but not DQ12 increased the gene expression of granulocyte–macrophage colony-stimulating factor (GMCSF) and granulocyte colony-stimulating factor (GCSF). These findings were confirmed by quantitative reverse transcriptase PCR. SD dose-dependently upregulated GMCSF and GCSF expression with significant 7 and 9-fold changes, respectively, at the highest tested concentration of 31 µg/cm2. Furthermore, we observed that SD significantly enhanced the secretion of GM-CSF and G-CSF by 2-fold. Both cytokines have previously been associated with lung diseases such as asthma and fibrosis. Hence, we present two molecular messengers that may contribute to the adverse health effects of desert dust and might serve as drug targets for this globally relevant non-anthropogenic air pollutant.

Knock down of APE1 suppressed gastric cancer metastasis via improving immune disorders caused by myeloid-derived suppressor cells

ABSTRACT Gastric cancer is a highly immunogenic malignancy. Immune tolerance facilitated by myeloid-derived suppressor cells (MDSCs) has been implicated in gastric cancer resistance mechanisms. The potential role of APE1 in regulating gastric cancer metastasis by targeting MDSCs remains uncertain. In this study, the plasmid Plxpsp-mGM-CSF was used to induce high expression of granulocyte-macrophage colony-stimulating factor (GM-CSF) in GES-1 cells. For tumor transplantation experiments, AGS, AGS+GM-CSF and AGS+GM-CSF-siAPE1 cell lines were established by transfection, followed by subcutaneous implantation of tumor cells. MDSCs, Treg cells, IgG, CD3 and CD8 levels were assessed. Transfection with siAPE1 significantly inhibited tumor growth compared to the AGS+GM-CSF group. APE1 gene knockdown modulated the immune system in gastric cancer mice, characterized by a decrease in MDSCs and an increase in Treg cells, IgG, CD3 and CD8. In addition, APE1 gene knockdown resulted in decreased levels of pro-MDSC cytokines (HGF, CCL5, IL-6, CCL12). Furthermore, APE1 gene knockdown inhibited proliferation, migration and invasion of AGS and MKN45 cells. AGS-GM-CSF cell transplantation increased MDSC levels and accelerated tumor growth, whereas APE1 knockdown reduced MDSC levels, inhibited tumor growth and attenuated inflammatory infiltration in gastric cancer tissues. Strategies targeting the APE1/MDSC axis offer a promising approach to the prevention and treatment of gastric cancer, providing new insights into its management.

Exploring the role of adipokines in exercise-induced inhibition of tumor growth

The integration of exercise prescriptions into cancer adjuvant therapy presents challenges stemming from the ambiguity surrounding the precise mechanism through which exercise intervention mitigates the risk of hepatocellular carcinoma (HCC) mortality and recurrence. Elucidation of this specific mechanism has substantial social and clinical implications. In this study, tumor-bearing mice engaged in voluntary wheel running exhibited a notable decrease in tumor growth, exceeding 30%. Microarray analysis revealed an upregulation of cytokine-related pathways as a potential explanation for this effect. The inclusion of granulocyte-macrophage colony-stimulating factor (GM-CSF) was found to enhance tumor cell proliferation, while the absence of GM-CSF resulted in a marked inhibition of tumor cell growth. The findings suggest that exercise-induced serum from mice can impede the proliferation of mouse tumor cells, with the adipokine chemerin inhibiting the growth factor GM-CSF. Additionally, exercise was found to stimulate chemerin secretion by brown adipose tissue. Chemerin suppression led to a reduction in the inhibition of tumor cell proliferation. The results of this study suggest that exercise may stimulate the release of adipokines from brown adipose tissue, transport them through the blood to the distant tumor microenvironment, and downregulate GM-CSF expression, alleviating tumor immunosuppression in the tumor microenvironment, thereby inhibiting at HCC progression. These findings provide a theoretical basis for incorporating exercise prescription into cancer treatment.

Expression of immune regulatory factors, chemokines and growth factorsin differentiated gastric cancer cells treated with an anticancer bioactive peptide combined with oxaliplatin.

Gastric cancer is one of the most common malignant tumors of the digestive system. An anticancer bioactive peptide (ACBP) was previously shown to have an important role in inhibiting the differentiation of the MKN-45, N87 and GES-1 cell lines. However, to date, research on the effects of inflammatory factors in MKN-45, N87 and GES-1 cell lines after treatment with ACBP combined with oxaliplatin (OXA) has not been performed. To investigate the expression of immune regulatory factors, tumor growth factors and chemotactic factors in differentiated gastric cancer cells treated with ACBP combined with OXA, the expression of cytokines, including interleukin (IL)-1β, IL-1 receptor antagonist, IL-2, IL-4, IL-6-10, IL-12, IL-13, IL-15, IL-17, Eotaxin, basic fibroblast growth factor (bFGF), granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon (IFN)-γ, monocyte chemoattractant protein (MCP)-1, IFN-γ-induced protein-10, macrophage inflammatory protein (MIP)-1α, platelet-derived growth factor (PDGF)-BB, MIP-1β, regulated upon activation, normal T cell expressed and presumably secreted, TNF-α and VEGF, was assessed with cell experiments using the Bio-Plex ProT Human Cytokine 27-plex Assay. The results indicated that immune regulatory factor, tumor growth factor and chemotactic factor expression levels were different after treatment with ACBP alone or ACBP combined with OXA. IFN-γ, IL-1β, IL-17, IL-9, IL-10, IL-15, bFGF, GM-CSF and PDGF-BB expression was decreased in MKN-45 and N87 cells after ACBP treatment (P<0.01) and ACBP+OXA treatment (P<0.01) compared with the control cells, which indicated that ACBP inhibited tumor growth by regulating these cytokines, and the combination treatment inhibited tumor growth by regulating these cytokines. MIP-1β, MCP-1 and IL-13 expression was decreased in MKN-45 and N87 cells after the combination treatment compared with ACBP treatment alone, which indicated that ACBP combined with OXA was able to inhibit tumor growth by regulating these cytokines, while the mechanism of action of the ACBP and OXA is actually different, e.g. for OXA, this would be to cause DNA damage response. Therefore, the ACBP and OXA combination treatment may be closely associated with tumor progression and metastasis with immunological competence by MCP-1, MIP-1β and IL-13 expression.

The Mechanistic Target of Rapamycin Complex 1 Pathway Contributes to the Anti-Tumor Effect of Granulocyte-Macrophage-Colony-Stimulating Factor-Producing T Helper Cells in Mouse Colorectal Cancer

ABSTRACT Introduction The role of granulocyte-macrophage-colony-stimulating factor-producing T helper (ThGM) cells in colorectal cancer (CRC) development remains unclear. This study characterizes the function of ThGM cells in mouse CRC. Methods Mouse CRC was induced by administrating azoxymethane and dextran sulfate sodium. The presence of ThGM cells in CRC tissues and the mechanistic target of rapamycin complex 1 (mTORC1) signaling in ThGM cells was detected by flow cytometry. The impact of mTORC1 signaling on ThGM cell function was determined by in vitro culture. The effect of ThGM cells on CRC development was evaluated by adoptive transfer assays. Results ThGM cells, which expressed granulocyte-macrophage-colony-stimulating factor (GM-CSF), accumulated in CRC tissues. mTORC1 signaling is activated in CRC ThGM cells. mTORC1 inhibition by rapamycin suppressed ThGM cell differentiation and proliferation and resulted in the death of differentiating ThGM cells. mTORC1 inhibition in already differentiated ThGM cells did not induce significant cell death but decreased the expression of GM-CSF, interleukin-2, and tumor necrosis factor-alpha while impeding cell proliferation. Furthermore, mTORC1 inhibition diminished the effect of ThGM cells on driving macrophage polarization toward the M1 type, as evidenced by lower expression of pro-inflammatory cytokines, major histocompatibility complex class II molecule, and CD80 in macrophages after co-culture with rapamycin-treated ThGM cells. Lentivirus-mediated knockdown/overexpression of regulatory-associated protein of mTOR (Raptor) confirmed the essential role of mTORC1 in ThGM cell differentiation and function. Adoptively transferred ThGM cells suppressed CRC growth whereas mTORC1 inhibition abolished this effect. Conclusion mTORC1 is essential for the anti-CRC activity of ThGM cells.

The novel role of activating receptor KIR2DS5 in preeclampsia

Preeclampsia (PE) is a serious complication of pregnancy. Decidual natural killer (dNK) cells were reported to participate in the remodeling of spiral arteries through producing a group of cytokines, including granulocyte–macrophage colony stimulating factor (GM-CSF). KIR2DS5 is an activating receptor of NK cells that specifically recognizes HLA-C2 on trophoblasts. Currently, there are no reports regarding the precise mechanism of KIR2DS5 in PE. This study included 30 PE patients and 30 healthy pregnant women. We found that the expressions of KIR2DS5 were significantly lower in PE deciduae compared to those of healthy pregnancies. By transfecting knockdown and overexpression lentivirus vectors of KIR2DS5 into dNK cells isolated from deciduae of early pregnancy, we altered the KIR2DS5 expression level in dNK cells. Then, these dNK cells and trophoblast cell lines were co-cultured as trophoblast-dNK cells. In the trophoblast-dNK cells, we examined the influence of KIR2DS5 on the biological manifestations of trophoblasts. As anticipated, overexpression of KIR2DS5 could facilitate cell proliferation, migration, and invasion. Furthermore, increased expression of KIR2DS5 inhibited cell apoptosis and enhanced the progression of cells from theG1 to theS stage. Further mechanistic study demonstrated a positive relationship between KIR2DS5 and GM-CSF in trophoblast-dNK cells. Accordingly, our observations indicated that a decrease in KIR2DS5 could reduce the expression of GM-CSF via the JAK2/STAT5 pathway, resulting in the failure of the activated signal to be transmitted to dNK cells and ultimately leading to the occurrence of PE. KIR2DS5 may be a new contributor for the prediction and diagnosis of PE.

The exploration of neuroinflammatory mechanism by which CRHR2 deficiency induced anxiety disorder

Inflammation stimulates the hypothalamic-pituitary adrenal (HPA) axis and triggers glial neuroinflammatory phenotypes, which reduces monoamine neurotransmitters by activating indoleamine 2,3-dioxygenase enzyme. These changes can induce psychiatric diseases, including anxiety. Corticotropin releasing hormone receptor 2 (CRHR2) in the HPA axis is involved in the etiology of anxiety. Omega(n)-3 polyunsaturated fatty acids (PUFAs) can attenuate anxiety through anti-inflammation and HPA axis modulation. However, the underlying molecular mechanism by CRHR2 modulates anxiety and its correlation with neuroinflammation remain unclear. Here, we first constructed a crhr2 zebrafish mutant line, and evaluated anxiety-like behaviors, gene expression associated with the HPA axis, neuroinflammatory response, neurotransmitters, and PUFAs profile in crhr2+/+ and crhr2−/− zebrafish. The crhr2 deficiency decreased cortisol levels and up-regulated crhr1 and down-regulated crhb, crhbp, ucn3l and proopiomelanocortin a (pomc a) in zebrafish. Interestingly, a significant increase in the neuroinflammatory markers, translocator protein (TSPO) and the activation of microglia M1 phenotype (CD11b) were found in crhr2−/− zebrafish. As a consequence, the expression of granulocyte-macrophage colony-stimulating factor, pro-inflammatory cytokines vascular endothelial growth factor, and astrocyte A1 phenotype c3 were up-regulated. While microglia anti-inflammatory phenotype (CD206), central anti-inflammatory cytokine interleukin-4, arginase-1, and transforming growth factor-β were downregulated. In parallel, crhr2-deficient zebrafish showed an upregulation of vdac1 protein, a TSPO ligand, and its downstream caspase-3. Furthermore, 5-HT/5-HIAA ratio was decreased and n-3 PUFAs deficiency was identified in crhr2−/− zebrafish. In conclusion, anxiety-like behavior displayed by crhr2-deficient zebrafish may be caused by the HPA axis dysfunction and enhanced neuroinflammation, which resulted in n-3 PUFAs and monoamine neurotransmitter reductions.

Soybean (Glycine max) INFOGEST Colonic Digests Attenuated Inflammatory Responses Based on Protein Profiles of Different Varieties.

Soybean compounds have been established to modulate inflammation, but less is known about how whole soybean compositions work together after digestion. The objective was to evaluate and compare the anti-inflammatory responses of different soybean varieties under simulated gastrointestinal digestion, with additional consideration of the glycinin:β-conglycinin ratio (GBR). Soybean colonic digests (SCD) inhibited cyclooxygenase (COX)-2 (25-82%), 5-lipoxidase (LOX) (18-35%), and inducible nitric oxide (iNOS) (8-61%). Varieties 88, GN3, and 93 were the most effective inhibitors. SCD (1 mg/mL) of varieties 81 and GN1 significantly (p < 0.05) reduced nitrite production by 44 and 47%, respectively, compared to lipopolysaccharide (LPS)-stimulated macrophages. SCD effectively reduced pro-inflammatory cytokine interleukin (IL)-6 (50 and 80% for 96 and GN1, respectively). Western blot results showed a decrease in the expression of iNOS, p65, and p50. The GBR was in the range of 0.05-1.57. Higher ratio correlated with higher production of IL-1β (r = 0.44) and tumor necrosis factor-alpha (TNF-α, r = 0.56). Inflammatory microarray results showed a significant decrease in expression of markers granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-6 in cells treated with GN1 SCD compared to LPS. The results suggested that SCD exerted its anti-inflammatory potential through nuclear factor kappa B (NF-κΒ) pathway inhibition by decreasing the levels of NF-κB-dependent cytokines and subunits, and inhibition of pro-inflammatory enzyme activity.

Decidual lymphatic endothelial cell-derived granulocyte-macrophage colony-stimulating factor induces M1 macrophage polarization via the NF-κB pathway in severe pre-eclampsia.

Direct interactions between macrophages and lymphatic vessels have been shown previously.In pre-eclampsia (PE), macrophages are dominantly polarized into a proinflammatory M1 phenotype and lymphangiogenesis is defective in the decidua.Here, we investigated whether decidual lymphatic endothelial cells (dLECs) affect macrophage polarization in PE. THP-1 macrophages were cocultured with dLECs or cultured in the conditioned medium (CM) of dLECs. Macrophage polarization was measured using flow cytometry. Granulocyte-macrophage colony-stimulating factor (GM-CSF) expression in dLECs was measured using qRT-PCR and ELISA. The activation of nuclear translocation of nuclear factor-κ (NF-κB), an upstream signaling molecule of GM-CSF, was assessed by immunocytochemical localization of p65. Through GM-CSF knockdown and NF-κB inhibition in dLEC, we evaluated whether the GM-CSF/NF-κB pathway of PE dLEC affects decidual macrophage polarization. The ratio of inflammatory M1 macrophages with HLA-DR+ /CD80+ markers significantly increased following coculturing with PE dLECs or culturing in PE dLEC CM, indicating that the PE dLEC-derived soluble factor acts in a paracrine manner. GM-CSF expression was significantly upregulated in PE dLECs. Recombinant human GM-CSF induced macrophage polarization toward an M1-like phenotype, whereas its knockdown in PE dLECs suppressed it, suggesting PE dLECs induce M1 macrophage polarization by secreting GM-CSF. The NF-κB p65 significantly increased in PE dLECs compared to the control, and pretreatment with an NF-κB inhibitor significantly suppressed GM-CSF production from PE dLECs. In PE, dLECs expressing high levels of GM-CSF via the NF-κB-dependent pathway play a role in inducing decidual M1 macrophage polarization.

The GM-CSF/CCL17 pathway in obesity-associated osteoarthritic pain and disease in mice

We have previously identified a granulocyte macrophage-colony stimulating factor (GM-CSF)/C-C motif ligand 17 (CCL17) pathway in monocytes/macrophages, in which GM-CSF regulates the formation of CCL17, and it is important for an experimental osteoarthritis (OA) model. We explore here additional OA models, including in the presence of obesity, such as a requirement for this pathway. The roles of GM-CSF, CCL17, CCR4, and CCL22 in various experimental OA models, including those incorporating obesity (eight-week high-fat diet), were investigated using gene-deficient male mice. Pain-like behavior and arthritis were assessed by relative static weight distribution and histology, respectively. Cell populations (flow cytometry) and cytokine messenger RNA (mRNA) expression (qPCR) in knee infrapatellar fat pad were analyzed. Human OA sera were collected for circulating CCL17 levels (ELISA) and OA knee synovial tissue for gene expression (qPCR). We present evidence that: i) GM-CSF, CCL17, and CCR4, but not CCL22, are required for the development of pain-like behavior and optimal disease in three experimental OA models, as well as for exacerbated OA development due to obesity, ii) obesity alone leads to spontaneous knee joint damage in a GM-CSF- and CCL17-dependent manner, and iii) in knee OA patients, early indications are that BMI correlates with a lower Oxford Knee Score (r=-0.458and p=0.0096), with elevated circulating CCL17 levels (r=0.2108and p=0.0153) and with elevated GM-CSF and CCL17 gene expression in OA synovial tissue. The above findings indicate that GM-CSF, CCL17, and CCR4 are involved in obesity-associated OA development, broadening their potential as targets for possible treatments for OA.

Th1 and Th2 epitopes of Cowdria polymorphic gene 1 of Ehrlichia ruminantium.

Cowdriapolymorphic gene 1 (cpg1, Erum2510, ERUM_RS01380) has been shown to induce 30% and 100% protection in sheep immunised by deoxyribonucleic acid (DNA) prime combined with DNA boost and DNA prime combined with protein boost, respectively, against heartwater infection via needle challenge. To localise its antigenic regions for inclusion in a multi-epitope DNA vaccine against heartwater, Erum2510 was cleaved into five overlapping subfragments. These subfragments were expressed individually in anEscherichia colihost expression system and evaluated for their ability to induce proliferative responses, Th1 and Th2 cytokines (interferon gamma [IFN-γ] and interleukin 4 [IL-4]) via enzyme-linked immunospot (ELISpot), quantitative real time polymerase chain reaction (qRT-PCR) and flow cytometry. Recombinant (r)proteins 3 and 4 were shown to induce immunodominant Th1 and Th2 immune responses characterised by the secretion of effector cytokines IFN-γ and IL-4 in addition to differential messenger ribonucleic acid (mRNA) expression of tumour necrosis factor (TNF), IL-2, IL-1, IL-18, IL-10, transforming growth factor (TGF), granulocyte-macrophage colony-stimulating factor (GM-CSF) and inducible nitric oxide synthase (iNOS). Thirty-seven overlapping synthetic peptides (16 mer) spanning the lengths of these immunodominantrproteins were synthesised and assayed. A peptide pool comprising p9 and p10 derived fromrprotein 3 induced a Th1-biased immune response. A peptide pool comprising p28 and p29 derived from rprotein 4 induced a mixed Th1 and Th2 immune response characterised by secretion of IFN-γ and differential mRNA expression of IL-1, IL-2, IL-10, IL-12, iNOS, TGF, TNF and GM-CSF. Only one of the peptides (p29) induced secretion of IL-4. Phenotypic analysis showed significant activation of cluster of differentiation 8+ (CD8+), cluster of differentiation 4+ (CD4+) and B+ lymphocyte populations. Findings suggest that Erum2510rproteins and synthetic peptides can induce both cellular and humoral immune responses, thereby implicating their importance in protection against heartwater.Contribution:This study will facilitate the design of an effective multi-epitope DNA vaccine against heartwater that will contribute to control this economically important disease in sub-Saharan Africa and beyond.

Nicotinamide N -methyltransferase promotes M2 macrophage polarization by IL6 and MDSC conversion by GM-CSF in gallbladder carcinoma.

Nicotinamide N -methyltransferase (NNMT), an enzyme responsible for the methylation of nicotinamide, is involved in many metabolic pathways in adipose tissue and the liver. However, the role of NNMT in editing the tumor immune microenvironment is not well understood. Here, we identified that NNMT can promote IL6 and granulocyte-macrophage colony-stimulating factor (GM-CSF) expression by decreasing the tri-methyl-histone H3 levels on the promoters of IL6 and CSF2 (encoding GM-CSF) and CCAAT/Enhancer Binding Protein, an essential transcription factor for IL6 expression, thus promoting differentiation of macrophages into M2 type tumor-associated macrophages and generation of myeloid-derived suppressor cells from peripheral blood mononuclear cells. Treatment of xenografted tumor models overexpressing NNMT gallbladder carcinoma (GBC) cells with the NNMT inhibitor JBSNF-000088 resulted in compromised tumor development and decreased expression levels of IL6, GM-CSF, tumor-associated macrophage marker CD206, and myeloid-derived suppressor cell marker CD33 but increased expression levels of CD8. In addition, elevated expression of NNMT in tumors of patients with GBC was correlated with increased expression levels of CD206 and CD33 but with decreased levels of CD8 and survival of patients. These data highlight the critical role of NNMT in GBC progression. Inhibition of NNMT by JBSNF-000088 is a potential molecular target for GBC immunotherapy.

Granulocyte-macrophage colony-stimulating factor: Conductor of the wound healing orchestra?

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a glycoprotein and is derived from both hemopoietic and nonhemopoietic sources which exert immunomodulatory properties. Various theories have been proposed to explain why some wounds become chronic and non-healing. Generalized suppression of inflammation locally or systemically may impede the body's physiological healing response by crippling the activity of reparative cells within the wound ecosystem. Thus, highlighting the importance of promoting host-directed therapeutics with immunomodulatory properties. The temporal and spatial expression of GM-CSF and GM-CSF receptors in the integumentary system suggests that epithelial-derived GM-CSF functions in an autocrine/paracrine manner. This may positively affect wound healing physiology via local inflammatory regulation promoting macrophage survival. Although diabetes negatively affects multiple aspects of wound healing GM-CSF activation is particularly impacted. Compared to acute/healthy wounds diabetic foot ulcers (DFU) only partially activate GM-CSF activity. There is a deleterious chain of events associated with this unfortunate sequala. DFUs also have a high proportion of monocytes and an absence of activated macrophages which results in an impaired inflammatory response. This may potentially serve as a vital point for GM-CSF to act as a companion diagnostic/theragnostic modality to help modulate the inflammatory response in wound healing. Correcting macrophage immune dysfunction with exogenous GM-CSF may help restore the immune balance in the wound ecosystem and jumpstart the wound healing cascade. Thus, the recognized beneficial role of GM-CSF in immune regulation across many studies provides a rationale for the initiation of the ongoing randomized controlled trials using GM-CSF.

Persistent diastolic dysfunction in chronically ischemic hearts following coronary artery bypass graft

ObjectiveA porcine model was used to study diastolic dysfunction in hibernating myocardium (HM) and recovery with coronary artery bypass surgery (CABG). MethodsHM was induced in Yorkshire–Landrace juvenile swine (n = 30) by placing a c-constrictor on left anterior descending artery causing chronic myocardial ischemia without infarction. At 12 weeks, animals developed the HM phenotype and were either killed humanely (HIB group; n = 11) or revascularized with CABG and allowed 4 weeks of recovery (HIB+CABG group; n = 19). Control pigs were matched for weight, age, and sex to the HIB group. Before the animals were killed humanely, cardiac magnetic resonance imaging (MRI) was done at rest and during a low-dose dobutamine infusion. Tissue was obtained for histologic and proinflammatory biomarker analyses. ResultsDiastolic peak filling rate was lower in HIB compared with control (5.4 ± 0.7 vs 6.7 ± 1.4 respectively, P = .002), with near recovery with CABG (6.3 ± 0.8, P = .06). Cardiac MRI confirmed preserved global systolic function in all groups. Histology confirmed there was no transmural infarction but showed interstitial fibrosis in the endomysium in both the HIB and HIB+CABG groups compared with normal myocardium. Alpha-smooth muscle actin stain identified increased myofibroblasts in HM that were less apparent post-CABG. Cytokine and proteomic studies in HM showed decreased peroxisome proliferator-activator receptor gamma coactivator 1-alpha (PGC1-α) expression but increased expression of granulocyte-macrophage colony-stimulating factor and nuclear factor kappa-light-chain enhancer of activated B cells (NFκB). Following CABG, PGC1-α and NFκB expression returned to control whereas granulocyte-macrophage colony-stimulating factor, tumor necrosis factor-α, and interferon gamma remained increased. ConclusionsIn porcine model of HM, increased NFκB expression, enhanced myofibroblasts, and collagen deposition along with decreased PGC1-α expression were observed, all of which tended toward normal with CABG. Estimates of impaired relaxation with MRI within HM during increased workload persisted despite CABG, suggesting a need for adjuvant therapies during revascularization.

Blocking DCIR mitigates colitis and prevents colorectal tumors by enhancing the GM-CSF-STAT5 pathway.

Dendritic cell immunoreceptor (DCIR; Clec4a2), a member of the C-type lectin receptor family, plays important roles in homeostasis of the immune and bone systems. However, the intestinal role of this molecule is unclear. Here, we show that dextran sodium sulfate (DSS)-induced colitis and azoxymethane-DSS-induced intestinal tumors are reduced in Clec4a2-/- mice independently of intestinal microbiota. STAT5 phosphorylation and expression of Csf2 and tight junction genes are enhanced, while Il17a and Cxcl2 are suppressed in the Clec4a2-/- mouse colon, which exhibits reduced infiltration of neutrophils and myeloid-derived suppressor cells. Granulocyte-macrophage colony-stimulating factor (GM-CSF) administration ameliorates DSS colitis associated with reduced Il17a and enhanced tight junction gene expression, whereas anti-GM-CSF exacerbates symptoms. Furthermore, anti-NA2, a ligand for DCIR, ameliorates colitis and prevents colorectal tumors. These observations indicate that blocking DCIR signaling ameliorates colitis and suppresses colonic tumors, suggesting DCIR as a possible target for the treatment of these diseases.

Peripheral γδ T Cells Regulate Neutrophil Expansion and Recruitment in Experimental Psoriatic Arthritis.

This study was undertaken to identify the mechanistic role of γδ T cells in the pathogenesis of experimental psoriatic arthritis (PsA). In this study, we performed interleukin-23 (IL-23) gene transfer in wild-type (WT) and T cell receptor δ-deficient (TCRδ-/- ) mice and conducted tissue phenotyping in the joint, skin, and nails to characterize the inflammatory infiltrate. We further performed detailed flow cytometry, immunofluorescence staining, RNA sequencing, T cell repertoire analysis, and in vitro T cell polarization assays to identify regulatory mechanisms of γδ T cells. We demonstrated that γδ T cells support systemic granulopoiesis, which is critical for murine PsA-like pathology. Briefly, γδ T cell ablation inhibited the expression of neutrophil chemokines CXCL1 and CXCL2 and neutrophil CD11b+Ly6G+ accumulation in the aforementioned PsA-related tissues. Although significantly reduced expression of granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-17A was detected systemically in TCRδ-/- mice, no GM-CSF+/IL-17A+ γδ T cells were detected locally in the inflamed skin or bone marrow in WT mice. Our data showed that nonresident γδ T cells regulate the expansion of an CD11b+Ly6G+ neutrophil population and their recruitment to joint and skin tissues, where they develop hallmark pathologic features of human PsA. Our findings do not support the notion that tissue-resident γδ T cells initiate the disease but demonstrate a novel role of γδ T cells in neutrophil regulation that can be exploited therapeutically in PsA patients.